Project Summary There are more than 250 million carriers of chronic hepatitis B virus (HBV) worldwide at risk of developing liver cirrhosis and hepatocellular carcinoma. To reduce this risk, two anti-HBV therapies are available: nucleoside analogs and interferon alpha (IFN?). Nucleoside analogs suppress viral replication but rarely lead to a cure due to the stability of the HBV genome as a covalently closed circular DNA (cccDNA). In contrast, IFN? cures approximately 10% of patients, but treatment has severe side effects and most patients don't benefit. Therefore, well-tolerated and more effective treatments are needed. Because IFN? can cure HBV, we sought to elucidate which interferon stimulated genes (ISGs) and pathways inhibit HBV. To this end, we screened a collection of ISGs and identified a potent HBV inhibitor. In addition, while conducting a genome-wide knockout screen for adenovirus, we identified a cellular protein that we later found was also necessary for HBV infection. Here we propose to characterize the mechanism of action of these two host factors and determine which cellular pathways affect HBV infection. Many aspects of the HBV lifecycle are not well understood due to the lack of robust in vitro and in vivo model systems. In recent years we have applied many of our liver systems and our expertise honed while studying hepatitis C virus to study hepatitis B virus. These efforts have laid the foundation for the proposed work and the model systems we will use while carrying out this work are a unique strength of the proposal. Overall, with our proposed work we will gain further insight into HBV biology and the host determinants of HBV infection. We hope this will uncover new strategies to cure chronic HBV infection.